Folate derivatives, useful in particular in the context of the folate assay

ABSTRACT

Use of a folate derivative to assay in vitro the folate in a sample such as a biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Nonprovisional patentapplication Ser. No. 14/655,258 entitled “FOLATE DERIVATIVES, USEFUL INPARTICULAR IN THE CONTEXT OF THE FOLATE ASSAY,” filed Jun. 24, 2015,which is a U.S. national stage filing under 35 U.S.C. § 371 ofInternational Application No. PCT/FR2013/053271 entitled “FOLATEDERIVATIVES, USEFUL IN PARTICULAR IN THE CONTEXT OF THE FOLATE ASSAY,”filed on Dec. 27, 2013, which claims priority from French PatentApplication Serial No. 1262898, filed Dec. 27, 2012. The contents ofeach of these applications are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

The present invention relates to folate derivatives, particularly usefulfor assaying a folate/folate(s) in an in vitro biological sample,preferably employing non-radioisotopic competition techniques.

STATE OF THE ART

Vitamin B9 is the generic denomination given to a very chemically andbiologically closely related family of compounds, all derived from folicacid. One of the main characteristics of these compounds is that theirpresence in insufficient quantity, or their actual absence, causesanaemia in man or in an animal. The folates family comprises, interalia, vitamin M, vitamin Bc, folacin and folic acid. Within the meaningof the present application, each of the compounds belonging to thisfamily is called “folate”, a plurality of members of this family iscalled “folates”, while the mixture of constituents of the vitamin B9family of is called “total folate”.

As is well-known to the man skilled in the art, folic acid, also calledpteroylmonoglutamic acid, is formed of a pterin group, a p-aminobenzoicgroup and a glutamate group as represented hereinafter by generalformula (G):

in which n is the integer 1.

As shown in this general formula, folic acid has two carboxylic acidfunctions on its glutamate part, one in the a position, the other in theγ position.

In food, folates are mostly in the form of reduced methyl- orformyl-polyglutamates. On digestion, these polyglutamates aretransformed into monoglutamates, actively absorbed by the enterocytes.Then, the monoglutamates are transformed into 5-methyltetrahydrofolate(5-MTHF), in which form the folates pass through the intestinal barrierand pass into the systemic circulation.

In blood, the major part of the circulating folates is bound with a weakaffinity to various proteins: α2-macroglobulin (40%), albumin (33%) andtransferrin (23%). Plasma concentrations of vitamin B9 vary from 5 to 15μg/L and are heavily impacted by food intake. The vitamin B9concentration is approximately 20 times higher in the red bloodcorpuscles which can contain up to 95% of the circulating folates.Multiple forms of folates are present in human serum, but thepreponderant circulating and intracellular form is5-methyltetrahydrofolate (5-MTHF), which is also the form of hepaticstorage. Generally, the biologically active compounds are solely thereduced forms: dihydrofolate (DHF), and mainly tetrahydrofolate (THF) aswell as its methyl or formyl derivatives. As indicated above, within themeaning of the present application, the denomination “folates” covers inparticular these reduced forms; each of said forms, taken separately,being called “folate”.

Eucaryotic cells, as well as certain procaryotic cells, are incapable ofsynthesising folic acid. They therefore use transmembrane transportsystems which allow internalisation of the exogenous molecule. At thepresent time, two main transport systems have been described. However,there probably also exist secondary routes such as passive diffusion.Oxidised folates like folic acid are transported inside cells by thefolate receptors (FR), “folate receptors” in the English language(Antony, 1992 [1]). These proteins were formerly called “folate bindingproteins” (FBPs). Three isoforms have been identified in man,respectively called FRα (P15328), FRB (P14207) and FRγ (P41439), thecode indicated between parentheses corresponding to the identifier ofthe protein in the UniProt database (http://www.uniprot.org). FRα and βare anchored in the plasmatic membrane by a lipid part,glycosylphosphatidylinositol (GPI). The γ isoform is secreted. Thereduced folates, for their part, are transported by a protein calledreduced folate carrier (P41440) or “reduced folate carrier” in theEnglish language (RFC). This is a highly glycosylated integral membraneprotein which has a plurality of transmembrane domains.

Due to their chemical structure, folates play an essential role in thesynthesis and metabolism of the basic constituents of our organism,namely amino acids and bases (purines and pyrimidines).

THF, an essential coenzyme, is capable of fixing and transferringradicals to a carbon atom. It is involved in the synthesis of glycineand the catabolism of histidine.

5-MTHF permits remethylation of homocysteine to methionine viamethylcobalamin and methionine synthase. Folates are also involved inseveral key steps of the biosyntheses of purines and pyrimidines, thusaffecting the synthesis of the nucleic acids DNA and RNA. Because ofthis central role, folate(s) deficiency has serious consequences andmany physiopathological expressions.

Severe folate deficiency gives rise to general, haematological andneuropsychiatric signs. Slowly, asthenia and anorexia appear. Anaemiacan be preceded by isolated macrocytosis. This anaemia, often ofmegaloblastic type, is one of the most frequent expressions of folatesdeficiency. In addition there is often a combined deficiency of folatesand of iron which results, instead of classic macrocytic anaemia, innormocytic anaemia with presence of Jolly bodies on the smear. Thisanaemia is due to the fact that purines and pyrimidines are notavailable in sufficient quantity, thus resulting in the impossibility ofblood stem cells synthesising genetic material and therefore dividing.Conversely, the existing cells continue to grow, which partiallyexplains the generally megaloblastic type of the anaemia associated withfolate deficiency.

Folates are also necessary for proper functioning of the brain, andcontribute to mental health and emotional balance. Thus, vitamin B9deficiency causes neuropsychiatric problems. These problems could inpart be linked to anomalies in the synthesis of certain amines andglycine. The latter is also a neurotransmitter.

Due to their contribution to the synthesis of genetic material, asatisfactory intake of folates is particularly necessary duringchildhood, adolescence and pregnancy. Indeed, psychomotor retardationand staturo-ponderal hypotrophy are often found in children havingfolates deficiencies. During pregnancy, a folate(s) deficiency can causedelay or anomalies in the development of the foetus, or even congenitaldeformities such as spina bifida which is incomplete closure of theneural tube, or even trisomy.

Folates deficiency is also associated with increased risk ofcardiovascular illnesses, more precisely arterial and/or venousthromboses and atheroscleroses. The risk is linked to the increase inthe plasmatic homocysteine level, resulting from lack of methylation ofthis compound into methionine.

This list is not limiting and folates deficiency can cause otherdisorders/pathologies. It is therefore of primary importance to be ableto assay all or part of the folates in a human or animal individual,preferably the “total” folate, i.e. formed by the mixture of thedifferent folate forms.

In addition, it is also important to be able to quantify all or part ofthe folates present in samples of food origin (intended for human oranimal consumption) in order to verify the vitamin B9 contribution ofthe foods in question. It may also prove advantageous to assay thefolates in products of food type, in order to ensure that these containa sufficient quantity/concentration of folate(s). Such food supplementscan in particular serve to prevent possible vitamin B9 deficiencies.

Assaying the folates in whole blood, serum, plasma, or in the red bloodcells has a certain advantage from a clinical point of view. Reductionin the blood folates concentration potentially results inexpresses adeficiency which should be clinically investigated, possibly inassociation with other vitamin or metabolite assays. The blood folateslevel is subject to variations depending on diet or the taking ofmedications. It is the folates level of red blood cells which gives thebest estimate of the folates reserves of the organism.

Several methods exist to permit the quantification of plasma, serumand/or red blood cell folates in biological samples of clinical origin,i.e. coming from patients. These methods can be classified in three maingroups, i.e.: (1) microbiological techniques, (2) chromatographictechniques and (3) competition immunoassays.

Microbiological techniques (1) generally use a “folates-dependent” seed,the growth of which is proportional to the vitamin level present in thesample to be assayed. Generally, the samples are deproteinised at 100°C. in the presence of vitamin C, which acts as an antioxidant. Contactwith the bacterial strain is effected for 20 hours at 37° C. The seedgerm most frequently used, Lactobacillus casei, is sensitive to all theoxidised and reduced forms of folates; other seeds are sensitive to morespecific forms. For example, Streptococcus faecalis permits the assay ofall forms of folates with the exception of 5-MTHF. The concentration of5-MTHF, the preponderant form in serum and red blood cells, is obtainedby the difference between the values of L. casei and of S. faecalis. Athird seed, Pediococcus cerevisiae, is sensitive exclusively toN5-formyl-THF (folinic acid).

These microbiological techniques (1), though generally sensitive andreproducible, are tedious and time-consuming. In addition, they presentrisks of interference with antibiotics and antimitotics, such asmethotrexate, trimethoprim and pyrimethamine.

Assays of chromatographic type (2) allow the separation of the differentcompounds belonging to the folates family. As examples ofchromatographic assay, can in particular be cited:

-   -   thin-layer chromatography assay coupled with HPLC (Reif, V. D.        et al., 1977 [2]),    -   chromatography assay (generally gas or liquid phase) coupled        with mass spectrometry (MS), for example by:        -   a) gas phase chromatography/mass spectrometry with isotope            dilution (ID-GCMS) (Dueker, S. R. et al., 2000 [3]), or        -   b) liquid phase chromatography-mass spectrometry in tandem            with isotope dilution (ID-LC-MS/MS) (Pfeiffer, C. M. et al.,            2004 [4]).

These assays of chromatographic type (2) notably have the disadvantageof requiring the development of very technical tests, requiringqualified personnel. In addition, the instrument proves to be expensive.

Having regard to the problems encountered on implementation of themicrobiological (1) and chromatographic (2) techniques (cf. above),competition immunoassays (3) have been developed. While reducing theanalysis time, these latter (3) permit the assay of “total” folate andtherefore the provision of a reliable clinical diagnosis relative to apossible folates deficiency.

These immunoassay processes (3), also called immunological assays orimmuno-chemical tests, involve the binding of the analyte to bedetected—in this case the folate(s)—with at least a first compound whichis a binding partner to this analyte. As the folate(s) assay is effectedby competition, the process also involves at least a second compoundwhich enters into competition with the folate to be assayed in relationto fixing on the binding partner, this second compound being a folatederivative. The monitoring of this reaction involves labelling one ofthe two compounds. This labelled compound is called labelled conjugateor tracer.

Of course, the prefix “immuno”, for example in “immunoassay”, is not tobe considered in the present application as strictly indicating that thebinding partner is an immunological partner, such as an antibody or anantibody fragment. Indeed, as is well-known to the man skilled in theart, this term is more widely used to designate tests and processes inwhich the binding partner, also called ligand, is not an immunologicalpartner but consists, for example, of a receptor of the analyte which isrequired to be assayed. The condition being that the binding partner iscapable of binding to the analyte, preferably in specific manner. Thus,it is known to use the term ELISA (Enzyme-Linked Immunosorbent Assay)for assays which use non-immunological binding partners stricto sensu,more widely called in English “Ligand Binding Assay”, which could betranslated into French as “Dosage utilisant la liaison à un ligand”,while the term “immuno” is included in the acronym ELISA. For the sakeof clarity and uniformity, the term “immuno” is used in the presentapplication to designate any assay using a suitable binding partner tobind to the analyte to be quantified, preferably in specific manner,even when this binding partner is not of an immunological nature ororigin in the strictest sense.

In the context of competition immunoassays (3), and when a labelledconjugate is used, three types of competition immunoassays aredistinguished depending on the nature of the labelled conjugate and onthe type of signal emitted by said conjugate, i.e.:

-   -   radio-isotopic immunoassays (Waxman S. and Schreiber C., 1980        [5]),    -   immuno-enzymatic assays or EIA “enzyme-linked        immunoassay-assay”; depending on the selected enzyme substrate,        the signal can be of colorimetric type (Hansen, S. I. and Holm        J., 1988 [6]) of fluorescence or chemiluminescent type,    -   electrochemiluminescent immunoassays (Owen, W. E. and        Roberts W. L. 2003 [7]).

The last two types of competition immunoassay are called“non-radioisotopic competition immunoassays”.

The development of the radioisotopic methods (RIA), from the 1960s,revolutionised the quantification of vitamins and in particular ofvitamin B9.

Patent application WO 80/00562 illustrates this by disclosingradioactive folate derivatives, substituted at the carboxylic acidfunction carried by the α carbon and/or by the γ carbon of the glutamatederivative. Radioactive labelling comes from the insertion of iodine-125or 130 in the phenol ring of a tyrosine structure.

French patent application FR-A-2455602 also relates to obtainingradioiodinated compounds and mentions pteroic acid derivatives ofgeneral formula:

in which the glutamate group is replaced by the radical X, this radicalX representing, as required, the residue of an amino acid or of a“des-carboxyamino acid” necessarily containing an aromatic orheterocyclic ring, indispensable for radioisotopic labelling (withiodine 125, 131 or 123). This iodinated aromatic or heterocyclic ring isseparated from the p-aminobenzoic acid group by a chain not includingmore than 5 carbon atoms, attached to the p-aminobenzoic group by asecondary amine. As “des-carboxyamino acid residue” containing anaromatic or heterocyclic ring, the following structures are disclosed:

However, these radioisotopic assays (RIA) had in particular thedisadvantage of the treatment of radioactive waste and the relativelyshort duration of the half-life of the labelled reagents.

This is why non-radioisotopic competition immunoassays have beendeveloped to the detriment of RIA which is only rarely used today.

By way of illustration, the publication Arcot J. et al, 2005 [8]describes a process for assaying folic acid by binding to a proteintagged with an enzyme (“enzyme-labelled protein binding assay” in theEnglish language), said labelled protein being FBP, and the processmethod being based on competition between the molecules of folic acidfree in the biological sample and those previously immobilised on amicrotitration plate to fix on the labelled FBPs. After rinsing, therevelation step is performed by introducing a colourless enzymesubstrate, causing blue colouration after cleaving by the enzyme fixedto the FBP. As is generally the case in competition assays, the quantityof free folic acid present in the sample is determined by reference to acalibration curve, from which the quantity of free folic acid present inthe sample is deduced as a function of the measured luminous intensity.

The Abbott Axsym Folate kit (Cat. No. B7K460, Abbott Laboratories) alsopermits the implementation of a competition folates assay process. As abinding partner, this kit uses the protein FBP and, as the labelledconjugate, pteroic acid (a folate analogue) bonded to alkalinephosphatase (ALPA). The principle of the test is based on competitionbetween the folic acid to be assayed and the above-mentioned conjugatein the context of fixing to the soluble FBP. Following the competitionreaction, the FBP is brought into contact with anti-FBP monoclonalantibodies and the antigen-antibody reaction takes place. Saidantibodies are bonded covalently to carboxymethylamylose, a polyanion.Thus, the complexes are captured by polyanion-polycation electrostaticinteractions, on a positively charged matrix. However, the Applicant hasdiscovered that the sensitivity of this assay was not completelysatisfactory, in particular regarding the quantification of low folatesconcentrations.

There is therefore an urgent need to improve the analytic sensitivity ofnon-radioisotopic folate immunoassays in order to obtain a processusable in samples of clinical origin (biological samples) and/or insamples of agri-food origin containing a low folate concentration.

STATEMENT OF THE INVENTION

The Applicant has, against all expectation, discovered novel folatederivatives which allow all or part of the disadvantages mentioned aboveto be remedied, in that their use in a non-radioisotopic competitionassay (such as an immunoassay), in particular as tracer, allows anincreased analytic sensitivity to be obtained, in particular in theranges of lowest folate concentration.

Thus, an object of the invention relates to the use of a folatederivative for assaying folate(s) in vitro in a sample, such as abiological sample, said assay being a non-radioisotopic competitionimmunoassay and said folate derivative being decarboxylated in the αposition. Said a position is such as shown in the general formula (G)mentioned above.

Indeed, the Applicant has in particular discovered, in surprisingmanner, that the folate derivatives decarboxylated in the α positionsignificantly improved the analytic sensitivity of non-radioisotopiccompetition immunoassays allowing the in vitro assay of folate(s).

Preferably, the folate derivative according to the invention isdifferent from the NSP-DMAE-HD-pteroate and NSP-DMAE-HEG-pteroatecompounds represented respectively by formulae (A) and (B) in claim 1.

Advantageously, the folate derivative according to the invention doesnot comprise an —(O—CH₂—CH₂)— structure and is different from theNSP-DMAE-HD-pteroate compound represented by formula (A) in claim 1.

As indicated above, the prefix “immuno”, for example in the term“immunoassay”, must not be narrowly interpreted as designating a bindingpartner of immunological nature and/or origin, such as an antibody.Indeed, the binding partner used in the competition immunological testcan be, for example, a receptor of the analyte which is required to beassayed, in this case the folates receptor. The non-radioisotopicimmunoassay techniques applicable according to the present invention canbe any techniques known to the man skilled in the art employing abinding partner binding with a sufficient affinity to the folate(s) forthe competition assay to be correctly performed.

According to a preferred embodiment, the folate derivative according tothe invention is used to assay a plurality of folates, or even,preferably, the total folate (as defined above).

According to a particular embodiment, the folate derivative according tothe invention is used to assay folic acid (pteroylmonoglutamic acid).

The terms “to assay” and “assaying” relate, in the present application,to the determination of a quantity/concentration of the analyte(s) inquestion, i.e. of the folate(s).

The invention also has as its object the use of a folate derivative toassay the folate in vitro in a sample such as a biological sample, saidassay being a competition immunoassay, preferably non-radioisotopic,said folate derivative responding to general formula (I):

-   -   in which:        -   X is an aliphatic hydrocarbon chain comprising 1 to 10            carbon atoms in which the carbon placed in the a position            does not carry an acyl function such as a carboxylic acid            function;        -   Y represents a functional group suitable to allow bonding to            a separate molecule M, such as a protein, said bonding            comprising the formation of at least one covalent bond            between Y and a functional group carried by said separate            molecule M.

The invention also has as its object the use of a folate derivative toassay the folate in vitro in a sample such as a biological sample, saidassay being a competition immunoassay, preferably non-radioisotopic,said folate derivative responding to general formula (I):

in which:

-   -   X is an aliphatic hydrocarbon chain containing from 1 to 10        carbon atoms;    -   Y represents a functional group suitable to allow bonding to a        separate molecule M, such as a protein, said bonding comprising        the formation of at least one covalent bond between Y and a        functional group carried by said separate molecule M.

By “aliphatic hydrocarbon chain” is understood, within the meaning ofthe present invention, a linear or open branched (acyclic) hydrocarbonchain. According to the definition commonly accepted and presented inthe reference works, a hydrocarbon chain must quite obviously beunderstood, within the meaning of the present invention, as onlycontaining hydrogen (H) and carbon (C). In other words the aliphatichydrocarbon chain X is solely functionalised by the functional group Ymentioned above and, by definition, does not contain a heteroatom (suchas oxygen, nitrogen, sulphur, phosphorus, halogens, etc.).

The combination of deacylation (of which decarboxylation is an example)in the a position of the folate derivative according to the inventionand of an aliphatic hydrocarbon chain comprising from 1 to 10 carbonatoms results in folate derivatives giving excellent analyticsensitivity when they are used in competition immunoassays (preferablynon-radioisotopic).

Said molecule M is, for example, a labelling molecule Mm. This moleculeM can also consist of a chemical arm or “linker”.

Preferably, X is a hydrocarbon chain comprising from 2 to 7 carbonatoms, preferably from 3 to 5 carbon atoms, advantageously X is ahydrocarbon chain of 3 carbon atoms or 5 carbon atoms.

Advantageously, X is a saturated hydrocarbon chain.

Preferably, X is a linear hydrocarbon chain.

According to a particularly preferred embodiment, X is a linear andsaturated aliphatic hydrocarbon chain, comprising a number of carbonatoms as defined above. In other words, and according to thisparticularly preferred embodiment, the folate derivative according tothe invention can be represented by the following general formula (I′):

in which:

-   -   n is an integer between 1 and 10. Advantageously, n is an        integer between 2 and 7, preferably between 3 and 5,        advantageously n represents the integer 3 or 5.    -   Y is as defined above.

According to a preferred embodiment, Y is is a group of electrophiliccentre type or a group of nucleophilic centre type, preferably a groupof electrophilic centre type, suitable to allow the formation of anamide, ester, or thioester bond, preferably amide or ester,advantageously amide, between Y and the functional group carried by saidseparate molecule.

According to a preferred embodiment, Y is a group of electrophiliccentre type, responding to the following general formula (II):

in which Gp is a leaving group, optionally bonded to the carbonylfunction by an L arm, Gp being suitable to be dissociated from the groupof electrophilic centre type in a reaction with a nucleophilic groupcarried by said separate molecule M, such as a primary amine.

The presence of the L arm is therefore optional in the compound ofgeneral formula (II), for which reason this L arm is shown betweenparentheses in said formula. Thus, for the purposes of the presentapplication, the group of formula (L)-Gp can therefore designate a groupof formula L-Gp (presence of the L arm) or a leaving group Gp (absenceof said L arm).

Preferably, said reaction of the group of electrophilic centre type ofgeneral formula (II) with said nucleophilic group carried by saidmolecule M—such as a primary amine—is a nucleophilic substitutionreaction.

Still in the preferred embodiment according to which Y is a group ofelectrophilic centre type, the (L)-Gp group is selected from groupssuitable to allow the formation of an amide, ester, or thioester bond,preferably amide or ester, advantageously amide, between said group ofelectrophilic centre type and a functional group carried by saidseparate molecule M. Preferably, the latter is a nucleophilic group suchas a primary amine.

Advantageously, the (L)-Gp group is selected from: —OH,—NH—(CH₂)_(m)—COOH, —N₃,

m being an integer between 1 and 10.

Among the above-mentioned (L)-Gp groups, the man skilled in the art willbe able to distinguish, without excessive difficulty, the Gp leavinggroups (such as the groups —OH, —N₃ and —N-oxy-succinimide) from theL-Gp groups, i.e. in particular:

in which the L arm is formed by the —NH—(CH₂)_(m)—CO (or—NH—(CH₂)_(m)—OC) part.

For the sake of clarity, it will be noted that the group called above“—N-oxy-succinimide” is the group of the following formula:

The use of such (L)-Gp groups favours the formation of an amide, ester,or thioester bond, preferably amide or ester, advantageously amide,between the group of electrophilic centre type Y and a functional groupcarried by the separate molecule M. These (L)-Gp groups are particularlysuitable to allow the formation of an amide bond between said group ofelectrophilic centre type and an amine function—such as a primaryamine—carried by the molecule M.

Preferably, m is between 1 and 5, advantageously between 1 and 3.

Advantageously, Y is a group of electrophilic centre type of generalformula (II) and the (L)-Gp group is selected from:

-   -   —OH and —NH—(CH₂)_(m)—COOH and

m being between 1 and 10 and preferably m being equal to 1.

According to a particularly preferred embodiment, Y is a group ofelectrophilic centre type of general formula (II) and (L)-Gp is the L-Gpgroup of the following formula:

In other words, the folate derivatives of the invention are thereforecharacterised by general formula (I) such as given above, in which X isas defined above and Y represents a bonding group activated or ready tobe activated to allow the formation of an amide, ester or thioesterbond, preferably amide or ester, advantageously amide, between saidderivative and a functional group carried by said separate molecule M.

According to a particularly preferred embodiment, Y is a bonding groupactivated or ready to be activated to allow the formation of an amidebond with a primary amine of said molecule M. In this embodiment, themolecules which can be bonded to the folate derivatives of the inventionare all molecules which naturally have a primary amine, such as aprotein, or any molecules which have been chemically modified to includesuch a primary amine, for example a modified biotin, having a primaryamine.

By “bonding group activated or ready to be activated”, is understood afunctional group suitable, where necessary after activation, to allowthe bonding of the folate derivatives of the invention to a functionalgroup carried by said separate molecule M (for example to a primaryamine carried by the latter).

According to a particularly preferred embodiment, Y is an activatedbonding group which allows the direct formation of an amide bond with afunctional group carried by the molecule M (for example a primaryamine), without this group needing to be previously modified. By way ofexample, the following groups can be cited:

m being an integer, preferably between 1 and 10, which constitutes anembodiment of the invention.

According to an alternative embodiment of the present invention, Y is abonding group ready to be activated, i.e. any group which must beactivated, by methods known to the man skilled in the art, to be capableof forming an amide, ester or thioester bond, preferably amide or ester,advantageously amide.

According to a particularly preferred embodiment, Y is a bonding groupready to be activated to form an amide bond between the folatederivative according to the invention and said molecule M. Such groupshave an —OH or —COOH group. As examples can be cited the groups —OH and—NH—(CH₂)_(m)—COOH; m being an integer, preferably between 1 and 10,which constitutes an embodiment of the invention.

According to a particular embodiment of the invention, m is between 1and 5, or between 1 and 3.

According to another embodiment, Y is selected from —OH,—NH—(CH₂)_(m)—COOH, and

m being between 1 and 10 and preferably being equal to 1.

According to another particularly preferred embodiment, the derivativeof general formula (I) is used in conjugate form with said molecule M,and said conjugate being represented by the following general formula(III):

-   -   in which X and M are as defined above, and in which Y′ is a        derivative of the functional group Y after bonding of the        derivative of general formula (I) to said molecule M,    -   said molecule M preferably being a labelling molecule Mm.

This conjugate of general formula (III) is different from the compoundNSP-TMAE-HD-pteroate represented by formula (A) in claim 1.

Preferably, Y′ is represented by the following general formula (IV):

-   -   or by the following general formula (V):

-   -   wherein R1 is —NH—, —O—, or —S—, preferably —NH— or —O—,        advantageously —NH—;    -   Y′ being preferably represented by general formula (IV).

When Y′ corresponds to general formula (IV), the conjugate according tothe invention can be represented by the following general formula(III′):

in which X, R₁ and M are as defined above.

According to a particularly preferred embodiment, R₁ is —NH— and formula(III′) is as follows:

in which X and M are as defined above.

According to a particularly preferred embodiment, still when Y′ is agroup of general formula (IV), X is —(CH₂)_(n)— and the conjugateaccording to the invention is represented by the following generalformula (III′):

in which M and n are as defined above. Thus, n is an integer between 1and 10. According to a preferred embodiment, n is an integer between 2and 7, preferably between 3 and 5, advantageously n is the integer 3 or5.

When Y′ corresponds to a group of general formula (still), the conjugateaccording to the invention is represented by general formula (III″):

in which X, R₁, and M are as defined above.

According to a preferred embodiment, R₁ is —NH— and the conjugateaccording to the invention then responds to the following formula(III″):

in which X and M are as defined above.

According to a particularly preferred embodiment, X is —(CH₂)_(n)— andthe conjugate according to the invention responds to the followinggeneral formula (III″):

in which M and n are as defined above. Thus n is an integer between 1and 10. According to a preferred embodiment, n is an integer between 2and 7, preferably between 3 and 5, advantageously n is the integer 3 or5.

Another object of the invention relates to a process for in vitro assayof the folate(s) in a sample, such as a biological sample, said assaybeing a non-radioisotopic competition immunoassay, said processcomprising the following steps:

-   a) bringing into contact, in said biological sample, (i) at least    one binding partner of the folate(s), such as an antibody suitable    to bind to the folate(s) or such as the folates receptor, with (ii)    at least one compound selected from a folate derivative such as    defined above and a conjugate according to the invention, at least    one of said compounds (i) and (ii) being suitable to emit a signal,-   b) optionally leaving a sufficient time lapse to allow the    competition reaction,-   c) measuring the intensity of the signal and deducing from it the    folate(s) concentration present in said biological sample by    reference to a calibration curve establishing a relationship between    measured signal intensity and folate(s) concentration.

This assay process can be implemented in a biological sample of clinicalorigin, i.e. taken from a human or animal patient. In addition, thisassay process also finds application in the assay of the folate(s) in asample of agri-food origin, such as foods intended for human or animalconsumption or food supplements. Generally, the assay process accordingto the invention is applicable whenever a folate(s) assay isrequired/necessary in a given sample.

Whatever the sample, of clinical origin or of food origin, the folatesassay requires a prior step of treatment of the sample in order todissociate the folates from other molecules present in these samples andwith which they interact. Such dissociation techniques are well known tothe man skilled in the art. By way of example, we can cite Europeanpatent EP 0382334 which discloses a method for preparation of serumsamples for assay of vitamin B12 and folates. U.S. Pat. No. 4,418,151discloses an alternative pre-treatment method. The manual of the AbbottAxsym Folate kit (Cat. No. B7K460, Abbott Laboratories) describes aprotocol for preparation of a haemolysate from a whole blood sample inorder to make all the folate molecules present in the red blood cellsaccessible to the assay.

The man skilled in the art, very well acquainted with non-radioisotopiccompetition immunoassay techniques will manage, without excessivedifficulty, to implement steps b) and c). In particular he will be fullycapable of constructing a calibration curve, on the basis of samplescontaining known concentrations of folate(s), thus establishing arelationship between the measured signal intensity and concentration offolate(s).

As mentioned above, competition immunological assay (also called“immunoassay by competition”) is an assay widely known to the manskilled in the art. It consists in assaying the analyte, here thefolate, in a sample in question, by creating competition between theanalyte of the sample and a derivative of this analyte, here the folatederivative according to the invention, with regard to fixing to abinding partner of immunological origin (for example an antibody or anantibody fragment) or otherwise (for example the folates receptor). Thebinding of the derivative of the analyte in question and of the bindingpartner is revealed by means of the presence of a tracer.

The derivative of the analyte (in this case the folate derivative) canbe used in the competition reaction, as indicated above, without priorbonding or after bonding to a marker to form a conjugate or tracer.

When the analyte derivative (in this case the folate derivative) is notbonded to a marker (in which case, it does not form the tracer but thecapture partner), the binding partner is then labelled to form thetracer of the reaction. When the analyte derivative (in this case thefolate derivative) is bonded to a marker (in this case a marker moleculeMm) to form a conjugate, the latter then constitutes the tracer and thebinding partner then becomes the capture partner.

The measured signal, emitted by the tracer, is then inverselyproportional to the quantity of folate(s) present in the sample to beassayed.

As binding partner of the folate(s), is used any molecule capable ofbinding to the folate(s). As examples of binding partner to thefolate(s), can be cited antibodies, antibody fragments, nanofitins, thefolates receptor or any other protein known to bond to the folate(s)with sufficient affinity to perform the non-radioisotopic competitionimmunoassay according to the invention.

When antibodies are used as binding partners, they can be, for example,polyclonal or monoclonal antibodies.

Polyclonal antibodies can be obtained by immunisation of an animal withthe target folate as immunogen, followed by recovery of the requiredantibodies in purified form, by taking serum from said animal andseparation of said antibodies from the other constituents of the serum,in particular by affinity chromatography on a column on which is fixedan antigen specifically recognised by the antibodies, in particular theimmunogen.

Monoclonal antibodies can be obtained by the hybridomas technique widelyknown to the man skilled in the art. The monoclonal antibodies can alsobe recombinant antibodies obtained by genetic engineering, by techniqueswell-known to the man skilled in the art.

As examples of antibody fragments can be cited the Fab, Fab′, F(ab′)₂fragments as well as the scFv (Single chain variable fragment), dsFv(Double-stranded variable fragment) chains. These functional fragmentscan in particular be obtained by genetic engineering.

Nanofitins (trade name) are small proteins which, like antibodies, arecapable of binding to a biological target thus permitting its detection,its capture or quite simply its targeting in an organism.

The binding partners used can be specific or not specific to thefolate(s). They are called specific when they are capable of bindingexclusively or virtually exclusively to the folate(s). They are callednon-specific when the binding selectivity to the folate(s) is low, andthey are capable of binding to other ligands, such as proteins orantibodies.

According to a preferred embodiment, at least one specific bindingpartner is used in the context of the non-radioisotopic competitionimmunoassay process according to the present invention.

When they are used in capture, the binding partners or the folatederivatives according to the invention can be bound or not bound to asupport by any technique known to the man skilled in the art.

The second step b) of the process of the invention is a conventionalstep of a competition immunoassay process.

The last step c) of the process according to the invention consists indetermining the folate(s) concentration. The measured signal isinversely proportional to the quantity of folate(s) in the sample. Inorder to determine the folate(s) concentration, the intensity of thesignal is measured and this intensity is plotted on a calibration curvepreviously obtained by techniques widely known to the man skilled in theart. Thus, for example, the calibration curve is obtained by performinga competition immunoassay using the same binding partner as well asincreasing quantities of folate(s). A curve is thus obtained by placing,for example, the folate(s) concentration on the abscissa and thecorresponding signal obtained after immunoassay on the ordonnate.

When compound (ii) is a conjugate according to the invention, asdescribed above, which constitutes an embodiment of the invention, thesignal is generated by the tagging labelling of the labelling moleculeMm, as described above.

When compound (ii) is a folate derivative of the invention, as describedabove, i.e. it is not bound to a marker molecule Mm, the signal consistsin the direct reading of the binding partner/folate derivative binding,which can in particular be performed by plasmon surface resonance or bycyclic voltammetry.

Preferably, compound (ii) is a conjugate according to the invention.

As indicated above, the folate derivatives and conjugates according tothe invention can be used to assay folate(s) in a sample. Preferably,these derivatives and conjugates are used to assay a plurality of (atleast two) folates, i.e. a plurality of folate forms (in particular thereduced forms) in said sample.

According to a particular embodiment of the invention, the folatederivatives and conjugates mentioned above are used to assay the totalfolate in a sample.

The invention also relates to an in vitro diagnostic method intended todetermine whether a human or animal patient, preferably human, has afolate(s) deficiency, said method comprising the following steps:

-   -   a) in a biological sample, preferably of whole blood, of plasma        or of serum, taken from said patient, assaying the folate(s) by        implementing the in vitro assay process according to the        invention, in order to deduce from it the folate(s)        concentration in the biological sample,    -   b) comparing said concentration with a threshold value,        corresponding to a predetermined folate(s) concentration below        which a patient is considered as deficient in folate(s), and    -   c) if the assayed concentration is less than said threshold        value, deducing from this that the patient has a folate(s)        deficiency.

Another object of the invention relates to a folate derivative ofgeneral formula (I)

-   -   in which:    -   X is as defined above;    -   Y is a group of electrophilic centre type or of nucleophilic        centre type, preferably a group of electrophilic centre type,        suitable to allow the formation of an amide, ester, or thioester        bond, preferably amide or ester, advantageously amide, between Y        and a functional group carried by a separate molecule M; and in        which:    -   when X is a linear and saturated hydrocarbon chain comprising a        number of carbon atoms equal to 2, Y is not an —(O—CH₂—CH₂)₂—NH₂        group;    -   when Y is a group of nucleophilic centre type consisting in a        primary amine, X comprises a number of carbon atoms different        from 6,    -   when Y is a group of electrophilic centre type, the latter        respond to the following general formula (II):

in which Gp is a leaving group, optionally connected to the carbonylfunction by an L arm, Gp being suitable to be dissociated from the groupof electrophilic centre type in a reaction with a nucleophilic groupcarried by said separate molecule M, such as a primary amine, and inwhich, when L is absent and Gp is —OH, X comprises a number of carbonatoms greater than 4.

According to a particular embodiment, the functional group Y does notcomprise an —(O—CH₂—CH₂)— structure.

This folate derivative of general formula (I) is suitable forimplementation of a non-radioisotopic competition immunoassay of thefolate(s) in a sample. It does not therefore include aradioisotope/radioelement.

Said folate derivative of general formula (I) as such is not labelled.Where necessary, said folate derivative will be labelled after bondingto a marker molecule Mm, said bonding comprising the formation of acovalent bond between the functional group Y of said folate derivativeand a functional group carried by said marker molecule Mm.

The folate derivative of general formula (I) is different from theNSP-DMAE-HD-pteroate compound represented by formula (A) in claim 1.

According to a particular embodiment, Y is different from group (C):

According to another particular embodiment, when X is a linear andsaturated hydrocarbon chain comprising a number of carbon atoms equal to2, Y is different from the following groups (D) and (E):

According to a modification of this “other particular embodiment”, Y isdifferent from the above-mentioned groups (D) and (E), and this whateverthe definition of X.

Preferably, said reaction of the group of electrophilic centre type ofgeneral formula (II) with said nucleophilic group carried by saidmolecule M—such as a primary amine—is a nucleophilic substitutionreaction.

According to a particular embodiment, when Y is a group of nucleophiliccentre type, the latter consists in a functional group different from aprimary amine.

According to a preferred embodiment, when Y is a group of electrophiliccentre type responding to general formula (II), (L)-Gp is a groupselected from: —OH, —NH—(CH₂)_(m)—COOH, —N₃,

-   -   m being an integer of between 1 and 10, preferably between 1 and        5, advantageously between 1 and 3, in which when L is absent and        Gp is —OH, X comprises a number of carbon atoms greater than 4.

Preferably, when Y is a group of electrophilic centre type of formula(II), (L)-Gp is selected from:

-   -   —OH and —NH—(CH₂)_(m)—COOH and,

-   -   m being between 1 and 10 and preferably m being equal to 1,    -   in which when L is absent and Gp is —OH, X comprises a number of        carbon atoms greater than 4.

According to a particularly advantageous embodiment, when Y is a groupof electrophilic centre type of formula (II), (L)-Gp is:

According to a particular embodiment, and still when Y is a group ofelectrophilic centre type of general formula (II), Gp is a groupdifferent from an —OH (hydroxyl group) group.

The invention also relates to a conjugate comprising a folate derivativeaccording to the invention and a separate molecule M, said derivativeand said molecule M being linked by at least one amide, ester, orthioester bond, preferably an amide or ester bond, advantageously anamide bond, between Y and a functional group carried by said molecule M.

According to a particularly preferred embodiment, said molecule M isselected from chemical arms or “linkers” or from the marker moleculesM_(m), said molecule M preferably being a marker molecule Mm permittingthe direct or indirect labelling of said folate derivative (the latterthen being called “tagged conjugate”).

Another object of the invention relates to a kit permitting theimplementation of the process according to the invention, said kitcomprising:

-   -   (i) at least one binding partner of said folate(s), such as an        antibody suitable to bind to the folate(s) or such as the        folates receptor,    -   (ii) at least one compound selected from a folate derivative        such as defined above and a conjugate according to the        invention, at least one of said compounds (i) and (ii) being        suitable to emit a signal, and    -   at least one calibration means.

Of course, this kit (also called “detection kit”) can comprise otherconstituents allowing or favouring the implementation of the immunoassayaccording to the invention, such as, for example, wash buffers and oneor more other reagent(s) allowing the labelling to be visualised, or theemission of a detectable signal.

The folate derivatives of the invention can be used in two differentmanners in the processes for assay of the folate(s) by competitionimmunoassay, such as diagnostic tests. Indeed, they are either used assuch, or they are used bonded to another molecule to form a conjugate.

Said “other molecule” is either a direct or indirect marker, or achemical arm or “linker”, or a chemical compound the bonding of which toa folate derivative has an advantage, in particular for implementationof a folate assay by competition immunoassay (preferablynon-radioisotopic).

Thus, the present invention also has as its object conjugates comprisingor formed of a folate derivative such as described above and of anothermolecule, in particular of a marker or of a chemical arm.

By marker, is understood any molecule capable of directly or indirectlygenerating a detectable signal. A non-limiting list of these directdetection markers consists in:

-   -   enzymes which produce a detectable signal for example by        colorimetry, fluorescence, luminescence, like horseradish        peroxidase, alkaline phosphatase, β-galactosidase,        glucose-6-phosphate dehydrogenase,    -   chromophores such as fluorescent, luminescent, colourant        compounds,    -   fluorescent molecules such as Alexa or phycocyanins,    -   electrochemiluminescent salts such as organometallic derivatives        based on acridinium all ruthenium.

Indirect detection systems can also be used, like for example ligandscapable of reacting with an anti-ligand. The ligand then corresponds tothe marker molecule Mm to constitute, with the folate derivativementioned above, the conjugate of the invention.

The ligand/anti-ligand pairs are well known to the man skilled in theart. By way of example can in particular be cited the following pairs:biotin/streptavidin, hapten/antibody, antigen/antibody,peptide/antibody, sugar/lectin, polynucleotide/complementarypolynucleotide.

The anti-ligand can then be directly detectable by the direct detectionmarkers described above or itself be detectable by anotherligand/anti-ligand pair, and so on.

These indirect detection systems can lead, under certain conditions, toamplification of the signal. This technique of amplification of thesignal is well known to the man skilled in the art, and reference can inparticular be made to prior patent applications FR98/10084 andWO-A-95/08000 of the Applicant.

Depending on the type of labelling used, the man skilled in the art willadd reagents permitting the labelling to be visualised or the emissionof a signal detectable by any appropriate type of measuring apparatus,like for example a spectrophotometer, a spectrofluorometer or a camera(for example a high-definition camera).

By chemical arm or “linker”, is understood any molecule able to bebonded to the derivative according to the present invention, saidmolecule being in addition capable of fixing onto a solid phase,covalently or non-covalently, in selective or non-selective manner.

As indicated above, the folate derivatives and the correspondingconjugates according to the present invention are particularly usefulfor in vitro determination of the folate(s) concentration in a sample ofclinical origin (for example a biological sample taken from a human oranimal patient) or a sample of agri-food origin (taken from a food orfrom a food supplement).

The determination of the folate(s) concentration using the derivative orconjugate according to the present invention may be performed in theculture supernatant or in the cellular lysate.

Another object of the invention relates to the process for obtaining aderivative according to the invention, said process comprising thefollowing steps:

-   a) synthesising, from pteroic acid, the compound of general formula    (VI):

-   -   in which W represents a group increasing the solubility of said        compound in organic solvent(s), such as a trifluoroacetyl group        (COCF3),

-   b) reacting said compound of formula (VI), obtained in step a), with    a compound of following general formula (VII):    NH₂—X—Y″—Z   (VII)    -   under conditions permitting the obtaining, by bonding, of the        compound of following general formula (VIII):

-   -   X being as defined above,    -   Z being an inert group, preferably alkyl or aryl, and    -   Y″ being a derivative of the Y group (such as defined above)        after bonding to said    -   Z group.

-   c) unprotecting, under conditions permitting this unprotection, the    compound of formula (VIII), in order to obtain the folate derivative    of formula (I) according to the invention.

According to a preferred embodiment, in step b), said compound offormula (VI), obtained in step a), is reacted with a compound offollowing general formula (VII):NH₂—X—Y″—Z   (VII)

-   -   in which Y″ is —C(O)O—,        in order to prepare a folate derivative of formula (I), in which        Y is —COOH.

In this preferred embodiment, if Z is a methyl or ethyl group, Y—Z isrespectively —C(O)O—CH3 or —C(O)O—CH3 in compounds of formulae (VII) and(VIII). If Z is an aryl group, then Y—Z is —C(O)O—Ar in said compounds(Ar symbolising this aryl group).

At the end of the unprotection step c), and still within this preferredembodiment, the above-mentioned group —C(O)O— results in a —COOH group.The folate derivative of general formula (I) according to the inventionthus obtained is called “acid” folate derivative. According to apreferred aspect of this embodiment, the process then comprises, afterstep c), the following additional step:

-   d) obtaining, from said acid folate derivative (in which Y=—COOH),    the NHS-folate derivative ester, i.e. the compound responding to the    following general formula (IX):

in which X is as defined above.

This compound of general formula (IX) represents one of the preferredfolate derivatives or the preferred folate derivative within the meaningof the present invention. Advantageously, X is a saturated linearaliphatic hydrocarbon chain comprising a number of carbon atoms between1 and 10, preferably between 2 and 7, advantageously between 3 and 5.

The methods for obtaining NHS (N-hydroxysuccinimides) esters are wellknown to the person skilled in the art. Examples of obtaining folatederivatives of formula (IX) according to the invention are presentedbelow (cf. examples 3 and 4 below) as illustrative and non-limitingexamples.

Pteroic acid is a commercially available compound. Unfortunately, thiscompound proves to have low solubility in organic solvents. It istherefore transformed, in step a), into a compound of general formula(VI) to facilitate the continuation of the present process.

Preferably, this compound of formula (VI) is N-trifluoroacetylpteroicacid (W=—COCF3). In this case, the pteroic acid is made to react,preferably, with trifluoroacetic anhydride, advantageously protected bynitrogen.

As indicated above, step b) is performed under reaction conditionsallowing the required reaction to be obtained. These reactionconditions, preferably, include the use of a bonding agent.

As indicated above, the inert group Z consists, for example, in a methylor ethyl group (in particular when Y″ is —C(O)O—, to give-C(O)O-Mp or—C(O)O-Et) or in an Fmoc group (in particular when Y″ is —NH—, togive-NH-Fmoc).

According to a particular embodiment, step b) comprises the followingtwo sub-steps:

b.1) forming, from the compound of formula (VI), obtained in step a), anactive intermediate such as N-trifluoroacetylpteroic acidisobutylformiate of the following general formula (VI′):

b. 2) reacting said active intermediate obtained in step b.1) with acompound of following general formula (VII):NH₂—X—Y″—Z   (VII)under conditions allowing the obtaining, by bonding, of the compound offollowing general formula (VIII):

in which W, X, Y″ and Z are as defined above.

According to a preferred embodiment, in steps b) and/or b.2) mentionedabove, the bonding is obtained by transamidification.

The unprotection step c) results in the elimination of the W group and,concurrently or subsequently, of the Z group, advantageously under theprotection of nitrogen.

This unprotection step c) is, preferably, performed in a basic medium,for example in a solution of NaOH. This basic solution allows theelimination of the W group and, concurrently, of the Z group (consistingfor example in a methyl or ethyl group), by saponification reaction.

DETAILED DESCRIPTION

The invention will be better understood by means of the followingexamples which are given in illustrative and non-limiting manner, withreference to FIGS. 1 and 2, in which:

FIG. 1 shows the synthesis steps of folate-C4-acid(4-(4-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methylamino)benzamido)butanoicacid) and NHS—C4-folate, and

FIG. 2 shows the synthesis steps of folate-C6-acid(6-(4-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methylamino)benzamido)hexanoicacid) and NHS—C6-folate.

EXAMPLE 1: PREPARATION OF4-(4-((2-AMINO-4-OXO-3,4-DIHYDROPTERIDIN-6-YL)METHYLAMINO)BENZAMIDO)BUTANOIC ACID (FOLATE-C4-ACID)

For the sake of clarity, the compound called “folate-C4-acid” is aderivative according to the invention, responding to the followinggeneral formula (I):

in which X is a linear and saturated aliphatic hydrocarbon chaincomprising 3 carbon atoms and Y is a group of electrophilic centre typeresponding to general formula (II):

in which L is absent and Gp is —OH.

1.1. Introduction

The reagents pteroic acid (CAS-Nr. 119-24-4), trifluoroacetic anhydride(CAS-Nr. 407-25-0), methyl 4-aminobutyrate (CAS-Nr. 3251-07-8), isobutylchloroformiate (CAS-Nr. 543-27-1) amino-6-hexanoic acid (CAS-Nr. 60-32-2also called 6-aminocaproic acid) and anhydrous dichloromethane (CAS-Nr.75-09-2) were obtained from Sigma-Aldrich.

At each synthesis step, high pressure liquid chromatography (HPLC) isused to monitor the reaction progress and for analysis of the products.The column used is a Vydac 218TP54, C18, 250×4.6 mm, 5 μm and the eluentis a gradient acetonitrile, water (0.1% trifluoroacetic acid) mixture.

1.2. Step 1: Obtaining N-Trifluoroacetylpteroic Acid

500 mg (1.60 mmole) of pteroic acid are introduced into a 50 mL flaskprovided with magnetic agitation, with an inlet and an outlet forprotection nitrogen. 10 mL of trifluoroacetic anhydride are added undernitrogen protection, drop by drop, over 30 minutes. The reaction mixtureis agitated at ambient temperature for 24 hours in the dark. Thereaction medium is evaporated under reduced pressure at ambienttemperature and the residue is dried under vacuum for 1 hour. Theproduct obtained is washed with 5 mL of ethyl ether and then dried undervacuum. The product is analysed by HPLC and used directly for thecontinuation of the synthesis.

1.3. Step 2: Obtaining N-Trifluoroacetyl-Folate-C4-Me

A mixture of 171 mg (0.42 mmole) of N-trifluoroacetylpteroic acid, 0.111mL of triethylamine (CAS-Nr. 121-44-8) and 2 mL of dry dimethylformamide(DMF, CAS-Nr. 68-12-2) is prepared and agitated at ambient temperatureunder nitrogen for 45 minutes to give medium No. 1. In another 10 mLflask, 150 mg (0.98 mmole) of methyl 4-aminobutyrate hydrochloride aremixed with 2 mL of dry DMF, and then 0.111 mL of triethylamine areadded. After agitation for 30 seconds, the mixture obtained is addedunder nitrogen protection to the prepared medium No. 1. The agitation ismaintained at ambient temperature for 3 hours and the reaction ismonitored by HPLC. The reaction medium is dried, without heat undervacuum. The residue is purified by silica gel 60 chromatography(0.040-0.063 mm, Merck Cat. No. 109385) with dichloromethane/methanoleluent, 5/1, v/v. 85 mg of product are obtained, which corresponds to a40% yield. The purity is 96%, determined by HPLC.

1.4. Step 3: Obtaining Folate-C4-Acid

85 mg (0.168 mmole) of prepared N-trifluoroacetyl-folate-C4-Me acid aremixed with 6 mL of methanol. 2 mL of a 1N NaOH mixture are added. Themixture is agitated at ambient temperature and in the dark for 16 hours.The reaction medium is neutralised at pH 2 with 50% of trifluoroaceticacid and then dried under reduced pressure at ambient temperature. Theresidue is washed with 10 mL of the demineralised water and dried undervacuum. 66 mg of folate-C4-acid are obtained, which corresponds to ayield of 98%. The purity is 97.2%, determined by HPLC.

The synthesis of folate-C4-acid is summarised in FIG. 1.

EXAMPLE 2: PREPARATION OF6-(4-((2-AMINO-4-OXO-3,4-DIHYDROPTERIDIN-6-YL)METHYLAMINO)BENZAMIDO)HEXANOIC ACID (FOLATE-C6-ACID)

The folate derivative called “folate-C6-acid” is a derivative accordingto the present invention responding to the following general formula(I):

-   -   in which X is a linear and saturated aliphatic hydrocarbon chain        comprising 5 carbon atoms, and in which Y is a group of        electrophilic centre type, responding to the following general        formula (II):

-   -   in which L is absent and Gp is —OH.

Folate-C6-acid(6-(4-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl-amino) benzamido)hexanoic acid) is synthesised in similar manner to folate-C4-acid. Inthe above-mentioned step 2, the methyl 4-aminobutyrate (NH₂—C4-Me) armis replaced by an ethyl 6-aminocaproate arm (NH₂—C6-Et, ethyl ester ofamino-6-hexanoic acid). This compound is prepared from amino-6-hexanoicacid in the presence of ethanol and acetyl chloride.

60 mg of folate-C6-acid are obtained from 171 mg ofN-trifluoroacetylpteroic acid, which corresponds to an overall yield of34%. The purity is 96%, determined by HPLC.

The synthesis of folate-C6-acid is summarised in FIG. 2.

Generally, the length of the hydrocarbon chain X in the derivative ofgeneral formula (I) can be varied by using, in step 2, alkylaminoalkanoate reagents of which the length of the hydrocarbon part ofthe alkanoate varies (4 carbon atoms to obtain the compound called“folate-C4-acid”; 6 carbon atoms to obtain the compound called“folate-C6-acid”).

EXAMPLE 3: PREPARATION OF THE ESTERS OF FOLATE-C4-NHS

The reagents, N-hydroxysuccinimide (NHS, CAS-Nr. 6066-82-6),1,3-dicyclohexylcarbodiimide (DCC, CAS-Nr. 538-75-0), dimethylsulphoxide(DMSO, CAS-Nr. 67-68-5) and tetrahydrofuran (CAS-Nr. 109-99-9) wereobtained from Sigma-Aldrich. 66 mg (0.166 mmole) of folate-C4-acidobtained in example 1, 28.8 mg (or 1.5×0.166 mmole) of NHS and 4 mL ofdry DMSO are introduced into a flask. 41.1 mg (or 1.2×0.166 mmole) ofDCC are introduced after 2 minutes of agitation. The agitation ismaintained at ambient temperature in the dark for 48 hours. HPLC is usedto monitor the progress of the activation. 7 mg of NHS and/or 4 mg ofDCC are added and the agitation is maintained for 72 additional hours.

The reaction mixture is filtered and the solution obtained is mixed with5 mL of dry tetrahydrofuran, then 170 mL of dry dichloromethane areadded. The mixture is centrifuged after 15 minutes without agitation torecover the precipitate which is then dried under reduced pressure,without heat and in the dark. 42 mg of folate-C4-NHS are obtained, whichcorresponds to a yield of 51%. The purity is 69%, determined by HPLC.

The obtaining of the folate-C4-NHS ester is represented in FIG. 1 (cf.last reaction step).

EXAMPLE 4: PREPARATION OF THE ESTERS OF FOLATE-C6-NHS

As for the folate-C4-NHS ester, the reagents used are the following:N-hydroxysuccinimide (NHS, CAS-Nr. 6066-82-6),1,3-dicyclohexylcarbodiimide (DCC, CAS-Nr. 538-75-0), dimethylsulphoxide(DMSO, CAS-Nr. 67-68-5) and tetrahydrofuran (CAS-Nr. 109-99-9), and wereobtained from Sigma-Aldrich.

60 mg (0.14 mmole) of folate-C6 acid obtained in example 2, a mixture ofDMF (2.5 mL) and DMSO (3 mL) dry solvents and 17.7 mg (1.1×0.14 mmole)of NHS are introduced into a flask. 32 mg (1.1×0.14 of DCC are addedafter 2 minutes of agitation. The agitation is maintained at ambienttemperature, in the dark, for 24 hours. HPLC is used to monitor theprogress of the activation. 30 mg of NHS and 30 mg of DCC are added andthe agitation is maintained for 96 additional hours.

The reaction medium is centrifuged for 3 minutes at 3000 rpm and therecovered liquid is mixed with 30 mL of the mixture of the solventsdichloromethane/petroleum ether (1/1) and then centrifuged again toobtain a yellow precipitate. The product is dried under reducedpressure, without heat and in the dark. 21 mg (29% yield) offolate-C6-NHS are obtained with an HPLC purity of 83.1%.

The synthesis of the folate-C6-NHS ester is summarised in FIG. 2 (cf.last reaction step).

EXAMPLE 5: PREPARATION OF THE FOLATE-C4-NHS-ALKALINE PHOSPHATASE ANDFOLATE-C6-NHS-ALKALINE PHOSPHATASE CONJUGATES

0.5 mL of a 20 mg/mL solution of recombinant alkaline phosphatase (ALP)(Roche, Ref. 03-535-452) are dialysed in Spectra/Por® tubing (cut-offlevel 6000-8000 Da, Spectrum Laboratories, USA) against 500 mL of 100 mMpH 8.3 carbonate buffer, under magnetic agitation, for one night, at+2/8° C. At the dialysis outlet, the concentration of the protein isdetermined by reading the optical density at 280 nm and thisconcentration is adjusted to 4 mg/mL.

The activated folate-C4-NHS and folate-C6-NHS esters obtained in example2 are again used in DMSO at concentrations of 0.39 mg/mL and 0.5 mg/mLrespectively, taking into account the purity.

For bonding of type (1-5) (one mole of alkaline phosphatase-5 moles offolate ester), 1.125 mL of the ALP solution are mixed with 256 μL of thefolate-C4-NHS ester solution on the one hand and 255 μL of thefolate-C6-NHS ester solution on the other. The percentage of DMSO in thereaction medium is 18.5%. The mixtures are incubated for one night at+2/8° C., under agitation on a wheel, in brown bottles.

Then, the reaction is stopped by addition of 10 mM lysine diluted inwater. The quantity of lysine added is equimolar with the quantity ofester used for bonding. Therefore 20.5 μL, of the lysine solution areadded for each of the bondings. The mixtures are incubated for 20minutes on a wheel, at +18/25° C.

After stopping the reaction, 1 mL of each of the conjugates are dialysedin Spectra/Por® tubing (cut-off level approximately 7000 Da) for 3 h at+18/25° C. against 500 mL of 50 mM Tris pH 7.4, 9 g/L NaCl, 0.9 g/Lazide buffer, under magnetic agitation. After 3 hours the tubes aretransferred into new baths again containing 500 mL of the same buffer.The dialysis is continued overnight at +2/8° C., under magneticagitation.

At the dialysis outlet, 10× conservation buffer (500 mM Tris pH 7.4, 90g/L NaCl, 50 mM MgCl₂, 1 mM ZnCl₂, 0.01% SDS, 9 g/L azide) is added tothe volumes recovered from the tubes. The volume obtained isapproximately 1 mL per conjugate. Following dialysis the conjugates areonly semi-purified: the dialysis permits elimination of the free,unreacted folate but not the free alkaline phosphatase. The conjugatescan be used in an immunoassay at this stage and this is what has hasbeen done in example 6 below.

To eliminate the free ALP and thus obtain conjugates with improvedpurity, hydrophobic interaction chromatography was performed using aRESOURCE Phenyl column (Cat No. 17-1186-01, GE Healthcare Lifesciences)mounted on an ÄKTA chromatography system. The flow rate of the pump isset to 2 mL/min. The TA buffer is 50 mM Tris pH 7.4, 9 g/L NaCl, 5 mMMgCl₂, 0.1 mM ZnCl₂, 0.9 g/L azide, 1.6 M (NH₄)₂SO₄. The TB buffer is 50mM Tris pH 7.4, 9 g/L NaCl, 5 mM MgCl₂, 0.1 mM ZnCl₂, 0.9 g/L azide. TheRESOURCE Phenyl column is equilibrated with TA buffer. The conjugate tobe purified is mixed volume for volume with the TB buffer. Then, 2volumes are added of the 50 mM Tris pH 7.4, 9 g/L NaCl, 5 mM MgCl₂, 0.1mM ZnCl₂, 0.9 g/L azide, 3.2 M (NH₄)₂SO₄ buffer. This step allows theconjugate to be in the TA buffer. The injection of the conjugate (628 μLfor folate-C4-NHS-ALP and 560 μL for folate-C6-NHS-ALP, in a 5 mL loop)is followed by a 20 mL wash in TA buffer. Then a 0 to 57% gradient of TBis applied for 30 mL, then a wash in 57% of TB buffer for 20 mL. Thisstep is followed by a second gradient of 57 to 100% of TB applied for 30mL, then by a wash in TB buffer for 20 mL. The last step consists in agradient of 0 to 100% in water for 20 mL, and then a wash in water for20 mL. The progress of the chromatography is monitored by measuring theoptical density at 280 nm. The fractions from 74 mL of elution up to 104mL (or in total 30 mL) are recovered, combined and then concentrated bydiafiltration using an Amicon cell (Amicon stirred cells, Millipore), anAmicon PM membrane with a cut-off level of 10 000 Da and the TB buffer.In this step, the volume of the conjugate solution is reduced toapproximately 0.5 mL. The conjugates are stored at +2/8° C. until theiruse in an immunoassay.

EXAMPLE 6: VITAMIN B9 ASSAY USING THE CONJUGATES FOLATE-C4-NHS-ALKALINEPHOSPHATASE AND FOLATE-C6-NHS-ALKALINE PHOSPHATASE AND COMPARISON WITHTHE AXSYM ASSAY CONJUGATE (ABBOTT LABORATORIES)

The immunological assays were performed using the VIDAS® automatedimmunoassay analysis system (bioMérieux). The single-use cone is usedboth as the solid phase for the reaction and as the pipetting system.The cartridge is composed of 10 wells covered with a sealed and labelledsheet of aluminium. The first well includes a previously cut-out part tofacilitate the introduction of the sample. The last well is an opticalcuvette in which the fluorescence of the substrate is measured. Thedifferent reagents necessary for the analysis are contained in theintermediate wells.

a) Sensitisation and Passivation of the Cones

The cones were sensitised with 300 μL of an “anti-folate bindingprotein” monoclonal mouse antibody (clone P8C5E4) diluted to 5 μg/mL ina 0.2 M tris, pH 6.2 buffer. After 6 hours of incubation at +18/25° C.,a wash is performed with a 1M solution of NaCl. Then, 300 μL of asolution of “folate binding protein” (FBP, Cat. No. F0524, ScrippsLaboratories) diluted to 6 ng/mL in a 100 mM, pH 7.4 NaCl 0.15 Mphosphate buffer containing human albumin and a sugar are added. Thesensitisation/passivation is continued at +18/25° C. overnight. Thecones are emptied, dried and then stored at +4° C. until use.

b) Preparation of a Range from Biological Samples

Human serum samples containing different folate concentrations wereobtained from the Biomnis laboratory (Lyon, France). The samples havingthe same concentrations were mixed in order to increase the availablevolume per range point. The mixtures were then aliquoted at 120 μL andfrozen at −20° C. until use. The nominal concentrations of each of thepoints are: 1.3 ng/mL-4.4 ng/mL-10 ng/mL-20 ng/mL. The range point 0ng/mL was prepared by dissolving 10% of human serum albumin in 10 mM pH8.5, 0.15 M NaCl phosphate borate buffer.

c) Extraction of the Samples

The objective of the extraction step is to dissociate the serum folatefrom its binding partners and to make it accessible for the assay. To250 μL of sample are added 50 μL of a 62.5 mg/mL TCEP(tris2-carboxyethyl)phosphine) solution and 215 μL of a solution of 0.8NNaOH+0.005% KCN. The mixture is incubated at +18/25° C. for 15 minutes,in the dark. After this stage, 1 mL of 1M pH4 glycine buffer is added.

d) Modus Operandi of the Immunoassay Reaction

The sample to be assayed (200 μL), extracted according to the protocoldescribed in c), is introduced into the first well of the cartridge.Then all the assay reaction steps are performed automatically by theVIDAS®. The cones prepared according to the protocol described in a) arewetted by a 1M pH 10, 0.1 M NaCl glycine and 2% saccharose buffer. Thesample to be assayed is mixed with 200 μL of a dilution of conjugatewhich is a folate derivative labelled with alkaline phosphatase. Thesample/conjugate mixture is incubated in the cone for approximately 20minutes during which competition takes place between the folates presentin the sample and the folate derivative of the conjugate for FBP proteinsites presented on the cone. Then, 3 successive washes with a 100 mMTris pH 7.4, 0.15M NaCl, 0.1% Tween® 20 buffer are performed in order toeliminate the non-fixed compounds. At the final revelation step, the4-methylombelliferyl phosphate substrate is aspirated and thendischarged into the cone; the enzyme of the conjugate catalyses thehydrolysis reaction of this substrate into 4-methylombelliferone, theemitted fluorescence of which is measured at 450 nm. The value of thefluorescence signal is inversely proportional to the folateconcentration present in the sample.

In the experiment presented in Table 1, three conjugates have beencompared. These are the two conjugates obtained in example 3 and, asreference, the conjugates used in the Abbot Axsym Folate kit (Cat. No.B7K460, Abbot Laboratories).

-   -   (i) the folate-C4-ALP and folate-C6-ALP conjugates were diluted        to a concentration of between 0.50-0.75 ng/mL in the conjugate        diluting buffer which contains 100 mM of Tris pH 8.5, 0.15 M        NaCl, 20 mg/L of mouse IgG, stabilisation agents, preserving        agents and other additives.    -   (ii) the conjugate of the folate Axsym kit is a conjugate of        pteroic acid (folate analogue) and alkaline phosphatase. This        conjugate was diluted to 1/80 with the conjugate diluting buffer        before use in the VIDAS®.

The range points prepared in b) were measured with each assay format.Table 1 below summarises the results obtained with the RFV (=relativefluorescence value) signal and B/B0% ratio. The B/B0% ratio is thesignal obtained for the range point tested divided by the signalobtained for the range point 0 ng/mL of folate, multiplied by 100.

TABLE 1 REF = Axsym conjugate Folate-C4-ALP Folate-C6-ALP [c] folateSignal Signal Signal (ng/mL) (RFV) B/B0% (RFV) B/B0% (RFV) B/B0% 0 3399100 3544 100 4118 100 1.3 3329 98 3095 87 3563 87 4.4 2442 72 2016 572619 64 10 925 27 995 28 1131 27 20 39 1 24 1 34 1

An 87% reduction in the signal is observed at 1.3 ng/mL of folate withthe folate-C4-ALP and folate-C6-ALP conjugates of the invention, whilewith the reference conjugate the signal reduction scarcely begins.

Consequently, the assays using the folate-C4-ALP and folate-C6-ALPconjugates are more sensitive than the assay using the referenceconjugate and permit better detection and quantification ofconcentrations less than 4.4 ng/mL, and even less than 1.3 ng/mL.

Regarding the folate derivatives and conjugates according to theinvention, in particular those responding to general formulae (I), (I′),(III), (III′), (III″), (VI), (VI′), (VIII) and (IX) mentioned above, itshould be noted that, even if the pterin part of these is showndiagrammatically in its ketone form, the present invention quiteobviously covers all—and each of the—tautomeric forms of said folatederivatives and conjugates able to be obtained at said pterin part, andin particular the 2-amino-4-hydroxy-6-methylpteridin form.

BIBLIOGRAPHICAL REFERENCES

-   1. Antony A C, The biological chemistry of folate receptors,    Department of Medicine, Indiana University School of Medicine,    Indianapolis, Blood. 1992 Jun. 1; 79(11):2807-20-   2. Reif V D, Reamer J T, Grady L T., Chromatic assays for folic    acid, J Pharm Sci. 1977 August:66(8):1112-6 PMID:894496-   3. Dueker S R, Lin Y, Jones A D, Mercer R, Fabbro E, Miller J W,    Green R, Clifford A J., Determination of blood folate using acid    extraction and internally standardized gas chromatography-mass    spectrometry detection, Anal Biochem. 2000 Aug. 1; 283(2):266-75    PMID:10906248-   4. Pfeiffer C M, Fazili Z, McCoy L, Zhang M, Gunter E W,    Determination of folate vitamers in human serum by    stable-isotope-dilution tandem mass spectrometry and comparison with    radioassay and microbiologic assay, Clin Chem. 2004 February;    50(2):423-32. Epub 2003 Dec. 11 PMID:14670827-   5. Waxman S. and Schreiber C., Determination of folate by use of    radioactive folate and binding proteins, Methods Enzymol. 1980;    66:468-83. No abstract available. PMID:7374487-   6. Hansen, S. I. and Holm, J., A competitive enzyme-linked ligand    sorbent assay (ELLSA) for quantitation of folates, Anal Biochem.    1988 July; 172(1):160-4. PMID:3189760-   7. Owen, W. E. and Roberts, W. L., Comparison of five automated    serum and whole blood folate assays, Am J Clin Pathol. 2003 July;    120(1):121-6. PMID:12866382-   8. Arcot J. and Shrestha A., Folate: methods of analysis, Food    Science and Technology, School of Chemical Engineering and    Industrial Chemistry, The University of New South Wales, Sydney,    Australia, 2005

The invention claimed is:
 1. A folate conjugate of general formula(III):

wherein X is a linear aliphatic saturated hydrocarbon chain containingfrom 2 to 10 carbon atoms; Y′ is one of the following general formula(IV) or (V):

wherein R₁ is —NH—, —O—, or —S—; M is a marker molecule capable ofdirectly or indirectly generating a detectable signal; and wherein theconjugate is not a compound of formula (A):


2. The conjugate according to claim 1, wherein X is a linear aliphaticsaturated hydrocarbon chain containing from 2 to 7 carbon atoms.
 3. Theconjugate according to claim 2, wherein the hydrocarbon chain containsfrom 3 to 5 carbon atoms.
 4. The conjugate of claim 1 wherein Y′ is ofgeneral formula (IV).
 5. The conjugate of claim 1 wherein R₁ is NH or O.6. The conjugate of claim 1 wherein R₁ is NH.
 7. The conjugate of claim1, said conjugate being selected from the group consisting of

wherein n is an integer between 2 and
 10. 8. The conjugate of claim 1wherein M is selected from an enzyme, a chromophore, a fluorescentmolecule or an electrochemiluminescent salt.
 9. The conjugate of claim 8wherein M is selected from the group consisting of horseradishperoxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphatedehydrogenase, Alexa, phycocyanins, and organometallic derivatives basedon acridinium and ruthenium.
 10. A kit comprising: (i) at least onebinding partner of folate(s) suitable to bind to the folate(s) or areceptor thereof; (ii) at least one folate conjugate corresponding togeneral formula (III):

wherein X is a linear aliphatic saturated hydrocarbon chain containingfrom 2 to 10 carbon atoms; Y′ is one of the following general formula(IV) or (V):

wherein R₁ is —NH—, —O—, or —S—; M is a marker molecule capable ofdirectly or indirectly generating a detectable signal; and wherein theconjugate is not a compound of formula (A):


11. The kit according to claim 10, wherein X is a linear aliphaticsaturated hydrocarbon chain containing from 2 to 7 carbon atoms.
 12. Thekit according to claim 11, wherein the hydrocarbon chain contains from 3to 5 carbon atoms.
 13. The kit of claim 10 wherein Y′ is of generalformula (IV).
 14. The kit of claim 10 wherein R₁ is —NH— or —O—.
 15. Thekit of claim 10 wherein R₁ is —NH—.
 16. The kit of claim 10, wherein thecompound of formula (III) is selected from the group consisting of

wherein n is an integer between 2 and
 10. 17. The kit of claim 10wherein M is selected from an enzyme, a chromophore, a fluorescentmolecule or an electrochemiluminescent salt.
 18. The kit of claim 17wherein M is selected from the group consisting of horseradishperoxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphatedehydrogenase, Alexa, phycocyanins, and organometallic derivatives basedon acridinium and ruthenium.